Bioconversion of short-chain hydrocarbons to fuels and chemicals

Abstract

An engineered microorganism(s) with novel pathways for the conversion of short-chain hydrocarbons to fuels and chemicals (e.g. carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives) is described. Key to this approach is the use of hydrocarbon activation enzymes able to overcome the high stability and low reactivity of hydrocarbon compounds through the cleavage of an inert C—H bond. Oxygen-dependent or oxygen-independent activation enzymes can be exploited for this purpose, which when combined with appropriate pathways for the conversion of activated hydrocarbons to key metabolic intermediates, enables the generation of product precursors that can subsequently be converted to desired compounds through established pathways. These novel engineered microorganism(s) provide a route for the production of fuels and chemicals from short chain hydrocarbons such as methane, ethane, propane, butane, and pentane.

We claim: 
     
       1. A method of producing a product comprising growing a genetically engineered bacteria in a culture broth containing a C1-C5 alkane and a terminal electron acceptor,
 wherein said genetically engineered bacteria converts the alkane to the product by oxygen-independent activation of the alkane, generating precursor intermediate acetyl-CoA, and producing a product from the acetyl-CoA, and 
 wherein the genetically engineered bacteria selected from the group consisting of  E. coli, Bacillus, Streptomyces,Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina , and  Methylococcus  comprises a vector expressing enzymes or overexpressing enzymes catalyzing: 
 a) a sequence of reactions for the oxygen-independent activation of the alkane via fumarate addition to a 2-methyl-alkyl-succinate and subsequent conversion of said 2-methyl-alkyl-succinate to an acyl-CoA; 
 b) a sequence of reactions for the generation of the product precursor acetyl-CoA and an acyl-CoA or keto-acid from said acyl-CoA; 
 c) a sequence of reactions for the regeneration of the fumarate through the conversion of said acyl-CoA or keto-acid to the fumarate; and 
 d) a sequence of reactions for the formation of the product from said product precursor acetyl-CoA; 
 wherein the alkane is the sole carbon source in the broth that can be activated; 
 wherein the enzymes comprise an alkyl succinate synthase that catalyzes the addition of the fumarate to the alkane to produce the 2-methyl-alkyl-succinate; 
 wherein one or more of the enzymes are overexpressed; and 
 wherein the method further comprises isolating the product formed in step d). 
 
     
     
       2. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA comprises:
 a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; 
 b. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; 
 c. an overexpressed 2-methyl-alkyl-malonyl-CoA mutase that catalyzes the isomerization of said 2-methyl-alkyl-succinyl-CoA to a 2-methyl-alkyl-malonyl-CoA; and 
 d. an overexpressed 2-methyl-alkyl-malonyl-CoA decarboxylase that catalyzes the decarboxylation of said 2-methyl-alkyl-malonyl-CoA to the acyl-CoA. 
 
     
     
       3. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA and generation of the product precursor acetyl-CoA and the acyl-CoA or the keto-acid comprises:
 a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; 
 b. an overexpressed 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; 
 c. an overexpressed 2-methyl-alkyl-succinyl-CoA dehydrogenase that catalyzes the conversion of said 2-methyl-alkyl-succinyl-CoA to 2-methyl-alkyl-2-butenoyl-CoA; 
 d. an overexpressed mesaconyl-C1-CoA-C4-CoA transferase that catalyzes the conversion of said 2-methyl-alkyl-2-butenoyl-CoA to 3-methyl-alkyl-2-butenoyl-CoA; 
 e. an overexpressed mesaconyl-C4-CoA hydratase that catalyzes the conversion of said 3-methyl-alkyl-2-butenoyl-CoA to 3-methyl-alkyl-3-hydroxy-succinyl-CoA; and 
 f. an overexpressed citramalyl-CoA lyase that catalyzes the conversion of said 3-methyl-alkyl-3-hydroxy-succinyl-CoA to the product precursor acetyl-CoA and the keto-acid. 
 
     
     
       4. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the oxygen-independent activation and conversion to the acyl-CoA and generation of the product precursor acetyl-CoA and the acyl-CoA comprises:
 a. the alkyl succinate synthase, wherein the alkyl succinate synthase is overexpressed; 
 b. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or 2-methyl-alkyl-succinyl-CoA synthetase that catalyzes the conversion of said 2-methyl-alkyl-succinate to a 2-methyl-alkyl-succinyl-CoA; 
 c. an overexpressed 2-methyl-alkyl-succinyl-CoA dehydrogenase that catalyzes the conversion of said 2-methyl-alkyl-succinyl-CoA to 2-methyl-alkyl-2-butenoyl-CoA; 
 d. an overexpressed mesaconyl-CoA hydratase/β-methylmalyl-CoA dehydratase that catalyzes the conversion of said 2-methyl-alkyl-2-butenoyl-CoA to 3-hydroxy-2-methyl-alkyl-succinyl-CoA; 
 e. an overexpressed β-methylmalyl-CoA lyase that catalyzes the conversion of said 3-hydroxy-2-methyl-alkyl-succinyl-CoA to glyoxylate and the acyl-CoA; 
 f. an overexpressed glyoxylate carboligase that catalyzes the conversion of said glyoxylate to tartronate semialdehyde; 
 g. an overexpressed tartronate semialdehyde reductase that catalyzes the conversion of said tartronate semialdehyde to D-glycerate; 
 h. an overexpressed glycerate kinase that catalyzes the conversion of said D-glycerate to 3-phospho-D-glycerate; 
 i. glycolytic enzymes that catalyze the conversion of said 3-phospho-D-glycerate to pyruvate, wherein the glycolytic enzymes are selected from the group consisting of phosphoglycerate mutase, enolase, and pyruvate kinase; and 
 j. a pyruvate formate lyase or pyruvate dehydrogenase that catalyze the conversion of said pyruvate to the acetyl-CoA. 
 
     
     
       5. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the generation of the product precursor acetyl-CoA and the acyl-CoA comprises:
 a. an overexpressed acyl-CoA dehydrogenase that catalyzes the conversion of said acyl-CoA to a transenoyl-CoA; 
 b. an overexpressed enoyl-CoA hydratase that catalyzes the hydration of said transenoyl-CoA to a 3-hydroxyacyl-CoA; 
 c. an overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the oxidation of said 3-hydroxyacyl-CoA to a ß-ketoacyl-CoA; and 
 d. an overexpressed thiolase that catalyzes the cleavage of the acetyl-CoA from said ß-ketoacyl-CoA to produce the acetyl-CoA and an acyl-CoA 2-carbons shorter than said acyl-CoA in step a. 
 
     
     
       6. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the regeneration of fumarate from the acyl-CoA or the keto-acid comprises:
 a. an overexpressed propionyl-CoA carboxylase that catalyzes the carboxylation of propionyl-CoA to (S)-methyl-malonyl-CoA; 
 b. an overexpressed methyl-malonyl-CoA epimerase that catalyzes the interconversion of said (S)-methyl-malonyl-CoA to (R)-methyl-malonyl-CoA; 
 c. an overexpressed methyl-malonyl-CoA mutase that catalyzes the isomerization of said (R)-methyl-malonyl-CoA to succinyl-CoA; 
 d. an overexpressed succinyl-CoA:2-methyl-alkyl-succinyl-CoA transferase or succinyl-CoA synthetase that catalyzes the conversion of said succinyl-CoA to succinate; and 
 e. an overexpressed succinate dehydrogenase that catalyzes the conversion of said succinate to fumarate. 
 
     
     
       7. The method of  claim 1 , wherein said pathway for the regeneration of the fumarate from the acyl-CoA or the keto-acid comprises:
 a. an overexpressed malate dehydrogenase for the conversion of said keto-acid to malate, wherein the keto-acid is pyruvate; and 
 b. an overexpressed fumarase for the dehydration of said malate to fumarate. 
 
     
     
       8. The method of  claim 1 , wherein said pathway for the regeneration of the fumarate from the acyl-CoA or the keto-acid comprises:
 a. an overexpressed carboxylic acid omega hydroxylase that catalyzes the conversion of said keto-acid to an omega-hydroxy-2-keto-acid; 
 b. an overexpressed alcohol dehydrogenase that catalyzes the conversion of said omega-hydroxy-2-keto acid to an omega-oxo-2-keto-acid; 
 c. an overexpressed aldehyde dehydrogenase that catalyzes the conversion of said omega-oxo-2-keto-acid to a dicarboxylic 2-keto-acid; 
 d. an overexpressed ketoreductase or malate dehydrogenase that catalyzes the conversion of said dicarboxylic 2-keto-acid to malate; and 
 e. an overexpressed fumarase for the dehydration of said malate to fumarate. 
 
     
     
       9. The method of  claim 1 , wherein said alkyl succinate synthase is encoded by  Azoarcus  sp. HxN1 masB,  Azoarcus  sp. HxN1 masC,  Azoarcus  sp. HxN1 masD,  Azoarcus  sp. HxN1 masE,  Azoarcus  sp. HxN1 masG,  Desulfosarcina  sp. BuS5 A39W_RS0101550 , Desulfosarcina  sp. BuS5 A39W_RS0101545 , Desulfosarcina  sp. BuS5 A39W_RS0101540 , Desulfosarcina  sp. BuS5 A39W_RS0101535 , Desulfosarcina  sp. BuS5 A39W_RS19630, or  Desulfosarcina  sp. BuS5 A39W_RS0101580. 
     
     
       10. The method of  claim 1 , wherein said enzymes catalyzing the sequence of reactions for the generation of the product precursor acetyl-CoA comprises:
 a. an overexpressed acyl-CoA dehydrogenase that catalyzes the conversion of said acyl-CoA to a transenoyl-CoA; 
 b. an overexpressed enoyl-CoA hydratase that catalyzes the hydration of said transenoyl-CoA to a 3-hydroxyacyl-CoA; 
 c. an overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the oxidation of said 3-hydroxyacyl-CoA to a ß-ketoacyl-CoA; and 
 d. an overexpressed thiolase that catalyzes the cleavage of the acetyl-CoA from said ß-ketoacyl-CoA to produce the acetyl-CoA and an acyl-CoA 2-carbons shorter than said starting acyl-CoA in step a. 
 
     
     
       11. The method of  claim 1 , wherein the sequence of reactions for the formation of the product from the product precursor acetyl-CoA is selected:
 a. a reverse beta oxidation (BOX-R) cycle comprised of:
 i. an overexpressed thiolase that catalyzes the non-decarboxylative condensation of an acyl-CoA primer with a 2-carbon donor acetyl-CoA to produce a ß-ketoacyl-CoA; 
 ii. an overexpressed 3-oxoacyl-[acyl-carrier-protein] reductase or overexpressed 3-hydroxyacyl-CoA dehydrogenase that catalyzes the reduction of a ß-ketoacyl-CoA to a ß-hydroxyacyl-CoA; 
 iii. an overexpressed 3-hydroxyacyl-[acyl-carrier-protein] dehydratase or an overexpressed enoyl-CoA hydratase or 3-hydroxyacyl-CoA dehydratase that catalyzes the dehydration of a (3R)-ß-hydroxyacyl-CoA to a transenoyl-CoA; 
 iv. an overexpressed enoyl-[acyl-carrier-protein] reductase or acyl-CoA dehydrogenase or trans-enoyl-CoA reductase that catalyzes the reduction of said transenoyl-CoA to an acyl-CoA that is two carbons longer than said acyl-CoA primer; and 
 v. an overexpressed termination pathway that catalyzes the conversion of an intermediate from said BOX-R cycle to said product; or 
 
 b. a fatty acid biosynthesis (FAS) pathway comprised of:
 i. an overexpressed acetyl-CoA carboxylase that catalyzes the conversion of acetyl-CoA to malonyl-CoA; 
 ii. an overexpressed malonyl-CoA-[acyl-carrier-protein] (“ACP”) transacylase that catalyzes the conversion of said malonyl-CoA to malonyl-ACP; 
 iii. an overexpressed β-ketoacyl-ACP synthase that catalyzes the decarboxylative condensation of said malonyl-ACP with an acyl-ACP primer to produce a ß-ketoacyl-ACP; 
 iv. an overexpressed 3-oxoacyl-ACP reductase that catalyzes the reduction of said ß-ketoacyl-ACP to a ß-hydroxyacyl-ACP; 
 v. an overexpressed 3-hydroxyacyl-ACP dehydratase that catalyzes the dehydration of a (3R)-ß-hydroxyacyl-ACP to a transenoyl-ACP; 
 vi. an overexpressed enoyl-ACP reductase that catalyzes the reduction of a transenoyl-ACP to an acyl-ACP that is two carbons longer than said acyl-ACP primer; and 
 vii. an overexpressed termination pathway that catalyzes the conversion of an intermediate from said FAS pathway to said product. 
 
 
     
     
       12. The method of  claim 11 , wherein said termination pathway is selected from the group consisting of:
 a. a CoA cleaving thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase, or an ACP cleaving thioesterase said product is selected from the group consisting of carboxylic acids, (3R)-β-hydroxy carboxylic acids, β-keto carboxylic acids, and α,β-unsaturated carboxylic acids; 
 b. an alcohol-forming coenzyme-A thioester reductase, an aldehyde-forming CoA thioester reductase and an alcohol dehydrogenase, an alcohol-forming ACP thioester reductase, or an aldehyde-forming ACP thioester reductase and an alcohol dehydrogenase and said product is selected from the group consisting of primary alcohols, 1,(3R)β diols, β-keto primary alcohols, and α,β-unsaturated primary alcohols; 
 c. an aldehyde-forming CoA or ACP thioester reductase and an aldehyde decarbonylase and said product is selected from the group consisting of linear alkanes, linear alkan-2-ols, linear methyl-ketones, and 1-alkenes; and 
 d. an aldehyde-forming CoA or ACP thioester reductase and a transaminase and said product is selected from the group consisting of primary amines, 3-hydroxy-amines, 3-keto-amines, and α,β-unsaturated primary amines. 
 
     
     
       13. The method of  claim 1 , wherein the terminal electron acceptor is SO 4   2− , NO 3   − , Fe 3+ , O 2 , or Mn 4+ .